Serum miRNAs differentially expressed in the MPM patients, compared to non-MPM affected controls.

<p>Light gray boxes indicate upregulated miRNAs. Dark grey boxes indicate downregulated/no change/undetectable miRNAs. Spark-line graph refers to the corresponding fold-change differential levels for each miRNA and for each individual patient. Column height in each patient sample, in the spark-line graph, was internally normalized, to show relative expression of various miRNAs within the same signature.</p

ICAM1 expression in endothelial cells treated with N-homocysteinylated albumin.

<p>Panel A: expression levels of ICAM1 transcripts quantitated by real time PCR (treated: 1 µmol/L homocysteinylated albumin; control: unmodified albumin); (p<0.001). Panel B: cytofluorimetric analysis of ICAM1 time course surface expression by EAhy926 endothelial cells treated with homocysteinylated albumin (C: unmodified albumin negative control; Tnf-α: positive control). Panel C: Time course of ICAM1 release in the culture medium, quantitated by ELISA assay. C: negative control (untreated cells); A: unmodified albumin; AH: 1 µmol/L homocysteinylated albumin; (p<0.001).</p

Validation by Q-PCR of transcriptome results relevant to upregulated gene involved in endothelial dysfunction.

<p>A: unmodified albumin; AH: homocysteinylated albumin. Gene expression in the AH sample group was significantly increased with respect to the corresponding genes in the A sample group (p<0.001).</p

KEGG pathways involving miRNA29, miRNA433 and miRNA25.

<p>Pathways involving gene targets for miRNA29, miRNA433 and miRNA25, as according to miRSystem software (ver. 20150312—mirsystem.cgm.ntu.edu.tw/). In order to draw inferences on potential functional interactions between miRNA and their gene targets, pathways identified are listed according to the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway map. Relevant nomenclature consists of a molecular network in terms of the KEGG Orthology (KO) groups. Genes are listed in each box according to their ability to serve as targets of each of the three miRNA considered in the upmost shaded headings. The miRNA targets involved in each specific pathway are reported according to a rank list where the first preferentially listed members, in each corresponding, box are common targets to more than one miRNAs.</p

VCAM1 expression in endothelial cells treated with N-homocysteinylated albumin.

<p>Panel A: Time course of induction of VCAM1 transcripts, in EAhy926 endothelial cells, by treatment with 1 µmol/L homocysteinylated albumin. Panel B: cytofluorimetric analysis of ICAM1 time course surface expression by EAhy926 endothelial cells treated with homocysteinylated albumin. (C: unmodified albumin negative control; Tnf-α: positive control). Panel C: Time course of ICAM1 release in the culture medium, quantitated by ELISA assay. C: negative control (untreated cells); A: unmodified albumin; AH: 1 µmol/L homocysteinylated albumin. (p<0.001).</p

Amplification conditions and primer pairs for PCR experiments.

<p>All conditions are relevant to real time PCR except for <i>VCAM1</i>, where tradition PCR has been employed. 35 amplification cycles have been performed.</p

Differential miRNA levels in the two outcome-related MPM patient groups.

<p>Fold change level for each miRNA is represented. Color code to each miRNA has been assigned in order to uniquely identify each miRNA within individual serum samples. Analyses were performed and calculation accomplished as described under “Material and Methods”. Bars, standard deviations derived from at least three different calculations.</p

Time-dependent, increased expression of ADAM17, MCP1 and Hsp60 in endothelial cells upon homocysteinylated albumin treatment.

<p>Panel A: Real time PCR evaluation during time course of <i>ADAM17</i>, <i>MCP1</i> and <i>Hsp60</i> mRNA. Panel B: ELISA assay of MCP1 released in the culture medium of treated cells. Panel C: Western blotting analysis of intracellular levels of ADAM17, and Hsp60, and analysis of Hsp60 released in the medium by immunoprecipitation and western blotting (Hsp60 IP). A: unmodified albumin control; AH: homocysteinylated albumin treatment. Levels of transcripts or proteins in the homocysteinylated albumin sample group were significantly increased compared to control (p<0.001).</p

Kaplan-Meier estimate of the overall survival time of the patients stratified for miRNA signature A or miRNA signature B.

<p>P-values were derived from a log rank test (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135331#sec002" target="_blank">Materials and Methods</a>”).</p

Effects of homocysteinylated albumin on monocyte adhesion.

<p>U937 monocytoid cells adhesion onto an endothelial monolayer (EAhy926) expressed as adherent cells (number/field; panel A) and percentage adherent cells compared to positive control (panel B). Counts are the mean of ten independent experiments, each carried out by counting five different fields/sample of triplicate samples. Examples of microscopic fields are shown on the right. C: negative control (untreated cells); A: unmodified albumin; AH: homocysteinylated albumin; AC: carboxymethylated albumin; T: positive control (Tnf-α). 0.3 or 1: homocysteine micromolar concentration present in the assay in the form of <i>N</i>-homocysteinyl groups bound to albumin, as comparable to the in vivo situation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031388#pone.0031388-Perna1" target="_blank">[14]</a>; p<0.0001.</p